PROJECT SUMMARY Diseases that arise from mutations in components of mitochondrial oxidative phosphorylation can be devastating, as mitochondria are crucial for energy synthesis. These diseases occur predominantly in infants and children, with a prevalence of 1 in 5000. Though virtually any organ can be affected, the heart is frequently involved, because cardiac function has such high energy requirements. These mitochondrial cardiomyopathies have a particularly grim prognosis, with mortality rates increased nearly three-fold compared to children without cardiac involvement. In linking cardiac function to mitochondrial metabolism, calcium signaling may be central to the pathological process. Calcium influx into the mitochondria can potently stimulate ATP synthesis, but excessive levels trigger mitochondrial failure and cell death. We hypothesize that, when oxidative phosphorylation becomes impaired, feedback regulation causes a compensatory increase in calcium influx, boosting ATP synthesis. However, after prolonged entry, mitochondrial calcium levels become excessive and trigger mitochondrial failure, exacerbating cardiac dysfunction. The rationale for this study is to determine whether such regulation exists in a well- characterized animal model of mitochondrial cardiomyopathies, which features genetic deletion of a mitochondrial transcription factor (Tfam) selectively in cardiomyocytes. The first aim is to determine whether the increased mitochondrial calcium levels found in preliminary studies are truly compensatory. For this aim, we will create animals with mitochondrial cardiomyopathies that have mitochondria that either cannot take up calcium, or are resistant to excessive calcium levels. The second objective is to determine the molecular mechanism causing enhanced mitochondrial calcium influx, and determine whether such enhancement can be replicated in cardiomyocytes derived from human induced pluripotent stem cells. In these analyses, we use an innovative set of techniques, including direct electrical measurement of mitochondrial calcium currents, that overcome technical challenges present in studying calcium transport. If successful, our research will define a significant new target for potential therapy in these devastating disorders.